N-(delta-(6-purinylthio)valeryl)amino acids and peptides

ABSTRACT

A COMPOUND OF THE GENERAL FORMULA:   PURIN-4-YL-S-(CH2)4-CO-NH-(CH2)N-CH(-Y)-CO-R   WHEREIN:   N IS AN INTEGER FROM 0 TO 5; R IS A MEMBER SELECTED FROM THE GROUP CONSISTING OF: (A) ALKOXY HAVING A STRAIGHT OR BRANCHED CHAIN OF 1 TO 8 CARBON ATOMS; (B) HYDROXY; (C) A RESIDUE OF A PEPTIDICALLY ATTACHED AMINO ACID; (D) A RESIDUE OF A DIPEPTIDE; THE SAID AMINO-ACID AND DIPEPTIDE RESIDUES BEING IN THE FORM OF THEIR FREE CARBOXY ACID; (E) A RESIDUE AS AT (C) AND (D) WHEREIN THE CARBOXY GROUP IS ESTERIFIED WITH AN ALCOHOL, HAVING A STRAIGHT OR BRANCHED CHAIN OF 1 TO 7 CARBON ATOMS; Y IS A MEMBER SELECTED FROM THE GROUP CONSISTING OF: HYDROGEN; ALKYL HAVING A STRAIGHT OR BRANCHED CARBON CHAIN OF 1 TO 5 ATOMS; PHENYL; 3-INDOLYL; -CH2OH, -CH2SH, -(CH2)SCH3 AND -(CH2)MCOR1 M IN THE LAST MEMBER BEING AN INTEGER FROM 1 TO 3 AND R1 BEING ALKOXY HAVING A STRAIGHT OR BRANCHED CHAIN OF 1 TO 8 CARBON ATOMS. AN EXAMPLE IS N-($-(6-PURINYLTHIO)VALERYL)GLYCINE ETHYL ESTER.   THE COMPOUNDS HAVE SHOWN AN ANTINEOPLASTIC EFFECT IN CERTAIN SPECIFIC TRANSPLANTABLE MICE TUMORS. THEY ARE DISTINGUISHED BY THEIR HIGH AFFINITY TO A PARTICULAR ORGAN OR TO THE TISSUE OF A PARTICULAR ORGAN AND THEIR ACUTE AND CHRONICAL TOXICITY IS LOW.

United States Patent O ABSTRACT OF THE DISCLOSURE A compound of the general formula:

s oH2)4ooNH oH2),.(i3Ho0R wherein n is an integer from to 5 R is a member selected from the group consisting of:

(a) alkoxy having a straight or branched chain of 1 to 8 carbon atoms;

(b) hydroxy;

(c) a residue of a peptidically attached amino acid;

((1) a residue of a dipeptide; the said amino-acid and dipeptide residues being in the form of their free carboxy acid;

(e) a residue as at (c) and ((1) wherein the carboxy group is esterified with an alcohol, having a straight or branched chain of 1 to 7 carbon atoms;

Y is a member selected from the group consisting of:

hydrogen;

alkyl having a straight or branched carbon chain of 1 to 5 atoms;

phenyl;

3-indolyl;

CH OH, -CH SH, -(CH )SCH and m in the last member being an integer from 1 to 3 and R being alkoxy having a straight or branched chain of 1 to 8 carbon atoms.

An example is N-[6-(6-purinylthio)valeryl]glycine ethyl ester.

The compounds have shown an antineoplastic effect in certain specific transplanta'ble mice tumors. They are distinguished by their high affinity to a particular organ or to the tissue of a particular organ and their acute and chronical toxicity is low.

BACKGROUND OF THE INVENTION Various cancerostatic agents are known and have been used clinically. Such agents are 6-(4-carboxybutyl)-thio purine, 6-mercaptopurine, and S-fluorouracil. However, some of these agents are quite toxic and some have only limited specificity for particular organs. Also, their effective action in the body may be limited in time as shown by radioactive tracing methods.

SUMMARY OF THE INVENTION It is therefore an object of the invention to provide for a cancerostatic agent that is of a highy specific action as to particular tumors or particular organs.

3,5h7,7fi5 Patented Mar. 2, 1971 It is a further object of the invention to provide such agents which have a lower toxicity than known compounds.

It is another object to provide agents of this kind which exhibit a prolonged time of activity as evidenced by radioactive tracing.

These objects are accomplished by the compounds of the invention which have the general formula:

l m N\ Y A T wherein:

n is an integer from 0 to 5, R is a member selected from the group consisting of:

(a) alkoxy having a straight or branched chain of 1 to 8 carbon atoms;

(b) hydroxy;

(c) a residue of a peptidically attached amino acid;

((1) a residue of a dipeptide; the said amino acid and dipeptide residues being in the form of their free carboxylic acid;

(e) a residue as at (c) and (d) wherein the carboxyl group is esterified with an alcohol, having a straight or branched chain of 1 to 7 carbon atoms;

Y is a member selected from the group consisting of:

hydrogen; alkyl having a straight or branched carbon chain of 1 to 5 atoms; phenyl; --CH OH, -CH SH, (CH )SCH and (CH COR m in the last member being an integer from 1 to 3 and R being alkoxy having a straight or branched chain of 1 to 8 carbon atoms.

The compounds of the invention have shown an antineoplastic effect in certain specific transplantable mice tumors.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The compounds of the invention may be obtained in various ways.

One convenient method starts from a derivative of 6- (4-carboxybutyl) thiopurine of the Formula II:

\NAN

wherein X is a chlorine atom or a N group.

This compound is condensed with an amino acid ester of the Formula III:

Y (III) wherein n and Y have the same meaning as in the Formula I, and R is alkoxy with a straight or branched carbon chain of 1 to 8 carbon atoms, to form a N-[6-(6- purinylthio)valeryl]amino acid ester of the Formula IV:

wherein n and Y have the same meaning as in Formula I and R has the same meaning as in Formula II.

The resulting product is then saponified with alkali. Alternatively it may be condensed once more, in succession, twice with an amino acid ester of the same type as shown in Formula III to form the corresponding ester either of the N-[-(6-purinylthio)valeryl]dipeptide, or of the N- [5- 6-purinylthio valeryl] tri peptide.

More specifically, the amino acid ester first obtained may be converted to the azide and may then be condensed with an amino acid ester of the general Formula III to form the corresponding ester of the dipeptide. The dipeptide may then be saponified by alkali to the free acid which latter thereupon is converted by another reaction with the amino acid ester in the presence of N,N-dicyclohexycarbodiimide to form the corresponding tripeptide ester.

The condensation of the 6-(4-carboxylbutyl)thiopurine derivative of Formula II with an amino acid ester of Formula III is preferably carried out if X in Formula II is chlorine in a medium of an inert organic solvent such as a halogenated aliphatic hydrocarbon having from 1 to 2 carbon atoms, preferably methylene chloride by using either at least two equivalents of the amino acid ester or by using one equivalent of an organic tertiary base, such as triethylamine.

When X in Formula II is a N group, the reaction is carried out in an inert organic solvent which may again be methylene chloride but may also be a solvent such as dimethylformamide or dioxane, using at least two equivalents of the amino acid ester or using again one equivalent of the ester with one equivalent of the organic base such as triethylamine.

In the alkali saponification of the N-[6-(6-purinylthio)- valeryl]amino acid ester of the Formula IV, and of the N-[5-(6-purinylthio)valeryl]dipeptide ester, there may be used an aqueous alkali metal hydroxide solution, e.g., of sodium or potassium hydroxide, in an amount of at least two equivalents in case of a monocarboxylic amino acid, and of at least three equivalents in case of a dicarboxylic amino acid ester. The reaction is carried out at a temperature of 025 C.

The reaction of the free N-[6-(6-purinylthio)valeryl] dipeptide acid with an amino acid ester of the Formula III, in the presence of N,N'-dicyclohexylcarbodiimide, to form the corresponding N-[6-(6-purinylthio)valeryl]tripeptide is preferably performed in the medium of an inert organic solvent, such as a halogenated aliphatic hydrocarbon having 12 carbon atoms, preferably methylene chloride.

The derivative of 6-(4-carboxybutyl) thiopurine of Formula II, e.g., the acid chloride which is used as the starting compound, is obtained preferably by reaction of a purine derivative with thionyl chloride in the medium of an inert organic solvent, e.g., methylene chloride, in the presence of dimethylformamide as catalyst. The condensation with the amino acid ester of Formula III can be carried out in the same medium without chloride isolation. Methylene chloride, as reaction medium, is useful also here because in preparing derivatives of optically active amino acids with it, no racemization takes place.

Another useful starting compound is the azide of 6-(4- carboxybutyl)thiopurine, which is easily available from the ester of the acid through the hydrazide thereof. The azide method is suitable also for the synthesis of N-[6- (6-purinylthio)valeryl]dipeptides, that is, by reaction of N-[5-(6-purinylthio)valeryl]amino acids (easily available from the corresponding hydrazides) with amino acid esters.

The following examples are given for purpose of illustration only and not with any intention of limiting the scope of the invention. The melting points of the examples have been determined in the Kofler block.

EXAMPLES (l) N-[fi-purinylthio)valeryJglycine ethyl ester. To a suspension of 25.2 g. (0.1 mole) 6-(4-carboxybutyl) thiopurine in 500 ml. anhydrous methylene chloride there were added 0.2 ml. of anhydrous dimethylformamide and upon stirring, 13 g. (0.11 mole) of thionyl chloride through a dropping device at laboratory temperatures. The reaction mixture was then heated under reflux for 2 hours with exclusion of atmospheric moisture. After cooling down, a solution of 41.2 g. (0.4 mole) of freshly redistilled glycine ethyl ester in 41 ml. methylene chloride is added in drops at a temperature of 5 to +5 C. After having stood overnight at the laboratory temperature, there were added 50 ml. of ethanol, whereupon the reaction mixture was shaken once with 50 ml. of a 1 M NaHCO and twice using 50 ml. water in each case. After the methylene chloride has been distilled oif, the crude N-[6-(6-purinylthio)valeryl] glycine ethyl ester (31 g.=92%) was crystallized out of aqueous ethanol under a water pump vacuum at 40-50; M.P. 155156 C.

(2) N [B-(purinylthio)valeryl]-L-leucine ethyl ester. To a suspension of 0.05 mole of 6-(4-carboxybutyl) thiopurine in 250 ml. anhydrous methylene chloride, prepared in the manner described in Example 1, a mixture of 8.76 g. (0.055 mole) of L-leucine ethyl ester and 11.2 g. (0.11 mole) of anhydrous triethyl amine, was dropwise added at 5 to +5 C., and the reaction mixture was left standing overnight at the laboratory temperature. The triethylamine hydrochloride eliminated was dissolved by addition of 25 ml. water, and the organic layer was processed in the way described in Example 1; the evaporation residue was crystallized with an acetone-hexane mixture. There was obtained 13.8 g. (70%) of N-[5-(6- purinylthio)valeryl]-L-leucine ethyl ester with M.P. 144- 145", [M 19.5 (c.=1, ethanol).

(3) N [5-(6-purinylthio)valeryl]-L-aspartic acid diethyl ester. Into a suspension of 0.05 mole of 6-(4-carboxybutyl)thiopurine in 250 ml. methylene chloride, prepared in the manner described in Example 1, 11.84 g. (0.0525 mole) of L-aspartic acid ethyl ester hydrochloride was introduced, and a solution of 16.7 g. (0.165 mole) of anhydrous triethylamine in 40 ml. methylene chloride was dropwise added with stirring at 5 to +5 C. After the reaction mixture had been processed in the manner cited in Example 2, the crude diester (10.0 g.) (95%) was crystallized from an acetone-hexane mixture. The N-[6-(6-purinylthio)valeryl]-L-aspartic acid diethyl ester obtained formed colorness needles with M.P. 114- 11) (C-=1, ethanol).

(4) N-[6 (6-purinylthio)valeryl]-L-glutamic acid diethyl ester. To a solution of 5.32 g. (0.02 mole) of 6-(4- carb0xybutyl)thiopurine hydrazine in 400 ml. of 0.1 M HCl, 20 ml. of 1 M NaNO were dropwise added upon stirring at 0 to +5 The stirring was continued at the same temperature until all reaction with iodine-starch test paper had disappeared (2 hours). The reaction mixture was then neutralized with 1 M NaHCO (20 ml.). The residue was eliminated, after 1 hour standing by strong suction at +5 and was washed with 20 ml. water. The still slightly damp azide (6 g.) was placed into 30 ml. methylene chloride. To the suspension 16.1 g. (0.08 mole) of L-glutarnic acid diethyl ester were added and the mixture was left to stand for 2 days at laboratory temperatures. On extracting the reaction mixture by shaking with water (10 ml.) and with 1 M NaHCO and evaporation of the solvent in a water pump vacuum, the

crude product crystallized out of an acetone-hexane mixt-ure. There were obtained 4.8 g. (55%) of the colorness N [6 (6-purinylthio)valeryl]-L-glutamic acid diethyl ester; M.P. 100-101, [u] 13.5 (c.=1, ethanol).

(5) N-[6-(6-purinylthio)valeryl]glycine. To a solution of 4.4 g. (0.11 mole) sodium hydroxide in 85 ml. water, 16.87 g. (0.05 mole) of N-[6-(6-purinylthio)valeryl]- glycine ethyl ester were added. After the material was dissolved the solution was left standing for 2 days at 20- 25 C. It then was acidified with dilute hydrochloric acid to pH 2-3, and the eliminated N-[6-(6-purinylthio) valeryl1glycine (15.1 g. 98%) was crystallized from water; M.P. 210-211.

(6) N-[6-(6-purinylthio)valeryl]-L-leucine. 1.97 g. (5 mmole) of N-[6-(6-purinylthio)valeryl]-L-leucine ethyl ester was placed upon stirring at to into a solution of 0.44 g. (11 mmole) sodium hydroxide in 20 ml. water. The mixture was left standing for 2 days at that temperature. After processing the mixture was described in Example 5, and recrystallizing the crude product from aqueous acetone, there were obtained 1.63 g. (90%) of N- [6-(purinylthio)valeryl] L leucine; M.P. 93-95 C. [a] 31 (c.=1, 0.1 M NaOH).

(7) N-[6-(6-purinylthio)valeryl]-L-aspartic acid, was was obtained by saponification of the N-[6-(6-purinylthio) valeryl]-L-aspartic acid diethyl ester (2.12 g., 5 mmole) with a solution of sodium hydroxide (0.66 g., 16 mmole) in water (21 ml.) at 0 to +5 (for 48 hours), or, alternatively, by the method of Example 6. The yield was 1.84 g. (100%) of N-[6-(purinylthio)valeryl]-L-aspartic acid; M.P. 198-199 (water), [u] +11".

(8) N [6 (6-purinylthio)valeryl]glycylglycine ethyl ester. To a solution of 3.23 g. (0.01 mole) of N-[6- (6-purinylthio)valeryl]glycine hydrazide in 200 ml. of 0.1 M HCl, ml. of a 0.1 M NaNO- solution was added dropwise upon stirring at 0 to +5. Stirring was continued at that temperature until no more reaction with iodinestarch test paper occurred (about 1 hour). The suspension of the acid azide hydrochloride obtained was neutralized with 10 ml. of 1 M NaHCO 100 g. of ammonium sulfate were then added and after one half an hour of stirring at 0 to +5 the azide was removed by strong suction, was washed with ml. water and transferred into ml. dimethylformamide. To the suspension 5.15 g. (0.05 mole) of freshly redistilled glycine ethyl ester were added, and the reaction mixture was left to stand for 2 days at the laboratory temperature. After distilling off the major part of the dimethylformamide by Water pump vacuum (50-60"), the evaporation residue was stirred up with 10 ml. water and the mixture adjusted with acetic acid to a pH of 6, the N-[6-(6-purinylthio)valeryl] glycylglycine ethyl ester thus eliminated (2.80 g., 71%) was crystallized from aqueous ethanol. It formed colorness needles; M.P. 227-229.

The N-[6-(6-purinylthio)valeryl] glycine hydrazide used as starting product was prepared in the following manner: 3.5 g. of N-[6-(6-purinylthio)valeryl]glycine ethyl ester was placed upon stirring at 20 into 17.5 ml. hydrazine hydrate. The solution was left standing for 2 days at the laboratory temperature. After distilling off the excess hydrazine hydrate by water pump vacuum, the evaporation residue was stirred up with 7 ml. water, the pH of the mixture was adjusted to 7 with dilute hydrochloric acid, and it was left to crystallize at +5 The N-[6-(6-purinylthio)valeryl]glycine hydrazide eliminated (3.05 g., 91%) was purified by crystallization from water; M.P. 231-233 (9) N [6 (6-purinylthio)valeryl]glycylglycyl glycine ethyl ester. To a suspension of 3.66 g. (0.01 mole) of N- [6-(6-purinylthio)valeryl1glycyl glycine in 36 ml. anhydrous methylene chloride, 1.24 g., (0.012 mole) of freshly redistilled glycine ethyl ester and thereafter a solution of 2.27 g. (0.011 mole) of N,N'-dicyclohexylcarbodiimide in 11 ml. methylene chloride were added, and the mixture left standing for 24 hours at 20. To the suspension thus obtained 50 m1. of a 90% aqueous ethanol and 1 ml. of

glacial acetic acid were added, and after 1 hours time the N,N'-dicyclohexyl urea formed was removed by suction, and the filtrate evaporated to dryness (40-50) by water pump vacuum. In order to remove the unreacted starting acid, the evaporation residue was stirred up with 25 ml. of 1 M NEIHCO3 solution for 1 hour, the undissolved portion was removed by suction and chromatographed on a silica gel column (100 g.), using a mixture of chloroform with 20% of methanol for elution. The fractions were collected of 25 ml. each. The first fractions (1-4) there chiefly contained the residual N,N'-dicyclohexyl urea, while in the further fractions (6th to 11th) there was the practically pure N-[6-(6-purinylthio) valeryl]glycylglycyl glycine ethyl ester (2.15 g., 48%). After recrystallization with 50% aqueous ethanol, the compound formed colorless needles with a M.P. of 23 8-240. By saponification according to Example 6, the N-[6-(6-purinylthio)va1eryl]glycylglycyl glycine, with a M.P. of 231-233 (water) was obtained.

In addition to the compounds prepared in the preceding Examples 1-9, there were prepared by the method of Examples 1-3, among others, the following N-[6-(6-purinylthio)valeryl]amino acid esters with a yield varying from 60 to 95% for the individual compound:

' (10) N-[6-(6-purinylthio)valeryl] glycine methyl ester, M.P. 156-158", (water).

(11) N-[6-(6-purinylthi0)valeryl] glycine isopropyl ester, M.P. 128-120", (acetone-hexane).

(12) N-[6-(6-purinylthio)valeryl] glycine n-butyl ester, M.P. 144-145, (acetone-hexane).

(13) N-[6-(6-purinylthio)valeryl]glycine n-octyl ester, M.P. 123-124", (acetone).

14) N- [6- 6-purinylthio valeryl] glycyl-DL-isoleucine, M.P. 163-165, (aqueous ethanol).

By the method of Example 8, among others, the following N- [6-(6-purinylthio)valeryl]dipeptide esters with yields varying from 60 to for the individual substances were prepared:

15) N- [6- 6-purinylthio valeryl] glycyl-L-leucine ethyl ester, M.P. 139-140 (acetone-hexane), [a] 8.7

(c.=1, pyridine).

(16) N [6 (6purinylthio)valeryl]glycyl-DL-isoleucine ethyl ester M.P. 141-143 (ethanol-hexane).

By the method of Examples 5-7, among others, the following N-[6-(6-purinylthio)valeryl]amino acids and dipeptides, in yields varying from to for the individual substances, were prepared:

(17) N-[6-(6-purinylthio)valeryl]-DL-a-alanine, M.P. 210-212, (water).

(18) N [6 (6-purinylthio)valeryl]-B-alanine, M.P. 171-173, (water).

19 N- [6- 6-purinylthi0 valeryl] -DL-B-phenylalanine, M.P. 113-115", (aqueous acetone).

(20) N [6 (6-purinylthio)valeryl]-DL-tryptophan, M.P. 123-125 (aqueous acetone).

21 N- [6- 6-purinylthio) valeryl] -DL-a-aminobutyric acid, M.P. 186-187", ('water).

(22) N [6 (6 purinylthio)valeryl]-w-aminocaproic acid, M.P. -156, (water).

(23) N [6 (6-purinylthio)valerylJ-DL-valine, M.P. 103-105 (aqueous ethanol).

(24) N-[6-(6-purinylthio)valeryl]-L-valine, M.P. 105- 107, (water), 11 (0.:1, 0.1M.NaOH).

(25) N-[6-(6-purinylthio)valeryl]-D-valine, M.P. 105- 107, (water), [6],; +11 (c.=1, 0.1M.NaOH).

(26) N-[6-(6-purinylthio)valeryl] -DL-norvaline, M.P. 178-180", (water).

(27) N [6 (6-purinylthio)valeryl]-DL-leucine, M.P. 108-110", (water).

(28) N [6 (6 purinylthio)valeryl]-DL-norleucine, M.P. -161, (water).

(29) N-[6-(6-purinylthio)valeryl] -DL-isoleucine, M.P. 103-105", (water).

(30) N-[6-(6 purinylthio)valeryl]-DL-glutamic acid, M.P. 211-213, (water).

(31) N-[6-(6 purinylthio)valeryl]-DL-w-aminoadipic acid, M.P. 195-197", (water).

(32) N-[6-(6 purinylthio)valeryll-DL-serine, M.P. 201-202, (water).

(33) N-[6-(6-purinylthio)valeryl]-fl-alanine ethyl ester, M.P. 132-133", (acetone).

(34) N-[-(6-purinylthio)valeryl]-DL {3 phenylalanine ethyl ester, M.P. 120-122", (acetone-hexane).

(35) N-[6-(6-purinylthio)valeryl]DL-tryptophan ethyl ester, M.P. 176-178, (acetone-hexane).

(36) N-[6-(6-purinylthio)valeryl]-DL-rx-aminobutyric acid, ethyl ester, M.P. 131-132", (acetone-hexane).

(37) N-[6-(6-purinylthio)valeryl]-w-aminocaproic acid ethyl ester, M.P. Ill-112, (aqueous acetone).

(38) N-[6 (6 purinylthio)valeryl]-DL-valine ethyl ester, M.P. 109-111", (acetone).

(39) N-[5-(6-purinylthiovaleryl]-L-valine ethyl ester, M.P. 114-115", (acetone-hexane), [a] 12,6 (c.=l, ethanol).

(40) N-[6-(6-purinylthio)valeryl]-D-valine ethyl ester, M.P. 114-115", (acetone-hexane), [a] |12,8 (c.=l, ethanol).

(41) N-[6-(6-purinylthio)valeryl]-DL-norvaline ethyl ester, M.P. 125-126", (ethanol-hexane).

(42) N-[6-(6 purinylthio)valeryl]-DL-leucine ethyl ester, M.P. 160-161", (acetone-hexane).

(43) N- [6- 6-purinylthio valeryl] -DL-norleucine ethyl ester, M.P. 116-117", (acetone-hexane).

(44) N-[5-(6-purinylthio)valeryl]-DL-isoleucine ethyl ester, M.P. 114l16, (acetone-hexane).

(45) N-[6-(6 purinylthio)valeryl]DL-glutamic acid diethyl ester, M.P. 111-113", (acetone-hexane).

(46) N-[6-(6 purinylthio)valeryl]-DL-a-aminoadipic acid diethyl ester, M.P. 92-93, (acetone-hexane).

(47) N-[E (6 purinylthio)valeryl]-DL-serine ethyl ester, M.P. 169-171", (aqueous ethanol).

(48) N-[6-(6 purinylthio)valeryl]-DL-,8-phenylserine ethyl ester, M.P. 174-175", (aqueous ethanol).

(49) N-[5-(6-purinylthio)valeryl] DL methionine ethyl ester, M.P. 125-126", (aqueous acetone).

(50) N-[6-(6 purinylthio)valeryl1-L-cystine diethylester, M.P. 105-107", (acetone), [a] 63 (c.=l, 90% aq. ethanol).

(51) N- [6- 6-purinylthio) valeryl] -DL-B-phenylserine, M.P. 120-122", (aqueous acetone).

(52) N-[6-(6-purinylthio)valeryl] DL methionine, M.P. 17l-172, (water).

(53) N,N' bis [6-(6-purinylthio)valeryl]-L-cystine, M.P. 153-155, (water), [a] 163 (c.=l, 0.1 M NaOH).

(5 4) N-[6-( 6-purinylthio valeryl] glycylglycine, 213-214", (water).

(55 N-[6 (6 purinylthio)valeryl]glycyl-L-leucine, M.P. 157-158", (water), [a] 9,5 (c.=1, 0,1 M NaOH).

The following table illustrates the pharmaceutical action of some of the compounds of the invention. The table shows a composition with three well known cancerostatics, which are 6-(4-carboxybutyl) thiopurine, 6-mercaptopurine, and 5-fiuorouracil.

Tumors of mice H: S180-Crocker sarcoma; SAK-the sarcoma originally induced by methylacridine; HK-milk gland adenocarcinoma; YYoshida ascitic reticulosarcoma of the Wister rat.

The antineoplastic action is expressed as a fraction, the numerator of which indicates the average tumor size in a treated group of ten animals in percent of the average tumor size in an untreated, equally large control group (:100% When the comparison with the control group (100/100) shows differences greater than 20%, Fischers probability coefficient (in the arrangement applied) assumes values below 12:0.05.

The substances tested were administered orally to mice at the optimum therapeutic doses established in 12 intervals during 24 hours, except that S-fluorouracil was applied subcutaneously. (In the table the doses in mg./kg. are quoted in parentheses.) The administration was started at the 3rd day after transplantation of the tumors S180, SAR, HK, and at the 2nd day for the Y growth. The tumor size was determined by weight after destruction of the animal.

The data given in the table show that the N-[6-(6-purinylthio)valeryl] glycine ethyl ester in mice with the Crocker sarcoma S180 is of a therapeutically highly specific action. It is both inhibiting of the tumor growth, and favorable to the life of the test animal. The analogous derivatives of glycyl glycine ethyl ester is of therapeutically specific action in mice with the SAK sarcoma, etc. (see the table). The N-[5-(6-purinylthio)valeryl]glycylglycyl glycine (not listed in the table) has significant therapeutical action in mice H with the ascitic sarcoma S37 (64/123) (with the same manner of application, dosage and test arrangement as used in the evaluation of the comparable derivative of the diglycine ester). On the other hand, the analogous derivatives of glycine ethyl ester and diglycine ethyl ester were practically ineffective in case of the S37 tumor.

As the table shows the substances of the invention are at least equally or even more active in animals with a particular tumor than the 6-mercaptopurine and 5- fiuorouracil used for comparison. The feature of the amino acid or peptide, or ester thereof, as the case may be, which are attached to 6 (4 carboxybuty1)thiopurine by a peptidic bond, are effective in some instances and also quite significant in other parameters of the physiological action. E.g. considering the chronic toxicity of the basal substance 6-(4-carboxybutyl) thiopurine (in mice LD about 12 g./kg. in oral application), it appears that the N-[8-(6-purinylthio)valeryl]-glycine is equally toxic with the same order of animals. However, the analogous diglycine ethyl ester derivative is under the same conditions substantially less toxic. Its chronic toxicity expressed in LD exceeds 50 g./kg. in per os application.

The mentioned glycine ester derivative, tagged with sulfur 8) when applied per os to an H mouse with tumor S 180 showed, within 3 hours after application, about a hundred times higher specific radioactivity in the pulmonary tissues, and about a three times higher specific radioactivity in the tumor tissues than 6-(4-carboxybutyl)thiopurine- S. The radioactivity concentration decreases rapidly in both tissues within lapse of time,

TABLE Tumor S180 SAK HK Y Compound:

N -[5-(G-puriuylthio)valcryllglyciuc ethyl ester. 56/147, (170) 60/95, (200) 93/117, (200) /109, (200) N-[6(6-purinylthio)valeryllglycylglycino ethyl estor 08/105, (200) 77/128, (200) 86/106, (200) /104, (200) N-[fi-(trpurinylthio)valcryl]DL-is0leuc1nc 93/85, (200) 97/98, (200) 84/119, (200) (200) N-[a-(opurinylthio)valeryllL-aspartic acid 108/99, (200) 91/99, (200) 85/103, (200) /130, (200) N-[6(6-purinylthio)valeryl]L-glutarnic acid diethyl estcr 81/120, (200) 79/96, (200) 113/113, (200) /89, (200) 6-(4-carboxybutyl)thiopurine 71/118, (200) 81/126, (200) 71 /124, (200) /158, (200) fi-mvrcnp op riue 68/123, (20) /118, (20) 68/121, 20) 4, 2 5-fluor0uracil 82/130, (25) 76/07, (25) 75/117, (25) /07, (25) and after 9 hours again is at a low level. In contrast with application per es of N-[6-(6-purinylthio)valeryl]- glycyl glycine- S to a mouse with ascitic tumor S37- 64/123, the radioactivity in pulmonary tissue reaches its maximum within 9 hours after administration, and after 13 hours is still at a high level. In case of a tumor tissue the maximum of specific radioactivity is reached within /2 hour of application whereupon the radioactive concentration decreases with time. However, after 24 hours it still is at about /5 of the maximum level.

What is claimed as new and desired to be protected by Letters Patent is set forth in the appended claims.

1. A compound of the formula:

wherein: n is or 1; R is a member selected from the group consisting of:

(a) n-alkoxy having 1 to 8 carbon atoms; (b) hydroxy;

(d) an alkyl ester of a residue as in (c), the alkyl moiety having from 1 to 7 carbon atoms;

Y is hydrogen, lower alkyl, or (CH CO R'; m is 1 or 2; and R is H or lower alkyl.

2. The compound of claim 1, which is N-[6-(6 purinylthio)valeryl] glycine ethyl ester.

3. The compound of claim 1, which is N-[6-(6 purinylthio)valeryl]-L-glutamic acid diethyl ester.

4. The compound of claim 1, which is N-[5-(6 purinylthio valeryl] -L-aspartic acid.

5. The compound of claim 1, which is N-[6-(6 purinylthio)valeryl] glycylglycine ethyl ester.

6. The compound of claim 1, which is N-[5-(6 purinylthio valeryl] glycylglycylglycine.

7. The compound of claim 1, which is N-[6-( 6 purinylthio) valeryl] glycylglycylglycine ethyl ester.

8. The compound of claim 1, which is N-[6-(6 purinylthio valeryl] -B-alanine ethyl ester.

9. The compound of claim 1, which is N- [5-(6 purinylthio valeryl] -DL-a-amin0butyric acid ethyl ester.

10. The compound of claim 1, which is N-[6-(6-purinylthio)valeryl] -D-valine ethyl ester.

11. The compound of claim 1, which is N-[5-(6-purinylthio)valeryl1-DL-norvaline ethyl ester.

12. The compound of claim 1, which is N-[6-(6-purinylthio)valeryl]-DL-leucine ethyl ester.

13. The compound of claim 1, which is N-[5-(6-purinylthio)valeryl]-DL-isoleucine ethyl ester.

14. The compound of claim 1, which is N-[6-(6-purinylthio)valeryl]-g1ycyl-L-leucine ethyl ester.

15. The compound of claim 1, which is N-[5-(6-purinylthio valeryl] -DL-isoleucine.

References Cited UNITED STATES PATENTS 2,524,422 10/1950 Boothe et a1. 260112.5 3,374,218 3/1968 Bcrnardi et a1. 260112.5 3,400,118 9/1968 Bodanszky et a1. 260-l12.5

OTHER REFERENCES Kaverzneva et al.: II, Izv. Akad. Nauk SSSR., Ser. Khim. 1966, 21482152.

Capek et al.: Coll. Czech. Chem-Commun. 30, 1030- 1035 (1965).

Kaverzneva et al.: Akad. Nauk SSSR., Izvestiia, Seriia Khimiia, 1966, 1l991203.

LEWIS GOTTS, Primary Examiner M. M. KASSENOFF, Assistant Examiner U.S. Cl. X.R. 

